Carbon and nitrogen apportioning in plants has a direct impact on their usefulness as agricultural commodities. Research directed toward altered regulation and reallocation of these assimilates represents a major effort in agricultural biology at both the biochemical and genetic levels. Several key enzymes in the metabolic pathways that direct carbon and nitrogen flow, process assimilates, and transfer the products into various sink tissues are of intense investigative interest. The biosynthesis of starch is one such well-regulated diurnal process (Smith, 1999; Preiss et al., 1998), with starch serving as a major carbon reserve as well as an energy source for plants and providing a major nutritional value of food crops. The dynamic throughput of plant carbon demands a tight, yet responsive, control of the key enzymes. To further add to the complexity, starch production occurs exclusively in membrane-bound plastids, thereby requiring import of the biosynthetic enzymes and regulators involved. However, localization within the plastid also serves to essentially distinguish enzymes directly involved in starch synthesis and therefore identify potential targets for genetic manipulation.
Starch is synthesized in leaves during the day from photosynthetically assimilated carbon derived via the reductive pentose phosphate pathway. One simple view of starch polymer production involves four types of enzymes: ADP-glucose pyrophosphorylase (AGP), starch synthases (SSs), starch-branching enzymes (SBEs), and starch-debranching enzymes (DBEs) (Smith, 1999). AGP forms ADP-glucose from glucose 1-phosphate. SSs add ADP-glucose to the elongating end of an α(1-4)-linked glucanchain, whereas SBEs cut α(1-4) links and rejoin them as α(1-6) branches that are subsequently trimmed by DBEs to yield short chains for further synthetic extension. However, different isoforms of SSs (soluble and granule-associated SSI, SSII, and SSIII) can participate in the production of branched glucans. For example, the granule-bound SSI, the waxy-encoded protein in maize, is directly and perhaps exclusively involved in producing amylose, an α(1-4) glucan polymer with little branching. In contrast, SSII participates in the synthesis of amylopectin, an α(1-6) branched glucan polymer that typically is found together with amylose to form starch granules. The ratio of these two glucans affects the physical characteristics of starch such as gelatinization and the absorption spectra of iodine-complexed starch. The alteration or absence of certain starch biosynthetic enzymes (Craig et al. 1998; Edwards et al. 1999; Lloyd et al. 1999) has a dramatic effect on the physical characteristics of starch, as well as the level of starch accumulated by the plant. In a similar, yet opposing, manner dark-regulated starch degradation occurs by means of catabolic enzymes such as amylase, α-glucosidase, and starch phosphorylase. The resulting starch stasis is the consequence of the metabolism and catabolism orchestrated by the respective enzymes.
Regulation of some enzymes involved in major resource allocation is affected by allosteric effectors, substrate levels, and product levels, as well as by phosphorylation (Sokolov et al., 1998; Sun et al., 1999; Imparl-Radosevich et al., 1999). For several key enzymes, regulation of activity is a two-step process involving phosphorylation of the enzyme, followed by formation of a complex with 14-3-3 proteins to complete the regulatory transition (Chung et al., 1999; Sehnke et al., 1997). For example, the assimilation of nitrogen for production of amino acids or nucleotide bases is tightly controlled by nitrate reductase (NR). NR responds to environmental signals, such as light and metabolite levels, by phosphorylation and interacts with 14-3-3 proteins (Bachmann et al, 1996; Moorhead et al., 1996), thereby rapidly altering nitrogen flux according to the plant's metabolic requirements. This phosphorylation-dependent interaction of NR with 14-3-3 proteins has become a paradigm for posttranslational regulation of metabolic enzymes (Chung et al., 1999). Recently, 14-3-3 proteins have been identified inside plastids (Sehnke et al., 2000), thereby implicating a potential role in starch regulation.
Several methods have previously been suggested for modifying the ability of plants to produce and store starch. See, e.g., U.S. Pat. No. 5,365,016; 5,498,831; 5,789,657; 5,792,920; 5,824,798; 5,830,724; 5,856,467; 5,959,180; 5,962,769; 5,981,852; 5,998,701; and 6,013,861.